Cell-extracellular matrix adhesion is a fundamental process that regulates cell shape, proliferation and differentiation. Abnormalities in cell-matrix adhesion and extracellular matrix assembly are closely associated with the pathogenesis of a variety of human diseases. The long-term objective of our research is to elucidate the mechanism by which cells regulate cell-matrix adhesion and extracellular matrix assembly. Integrin-linked kinase (ILK) is an important regulator of cell-matrix adhesion and fibronectin matrix assembly. This research project focuses on the molecular mechanism by which ILK functions in these processes. Our hypotheses are that (1) a multi-protein complex comprising ILK, PINCH and CH-ILKBP provides an important physical connection between cell adhesion receptors and the actin cytoskeleton at the cell-matrix contact sites and (2) a PINCH-related protein (PINCH-RP), which was recently cloned by the applicant, regulates the assembly of the PINCH/ILK/CH-ILKBP complex and thereby participates in the regulation of cell adhesion, actin cytoskeleton organization and matrix assembly. The proposed studies are designed to critically test these hypotheses. First, the sites of PINCH, ILK and CH-ILKBP that are involved in the assembly and the localization of the PINCH/ILKICH-ILKBP complex to cell-matrix contact sites will be defined by site-directed mutagenesis. Second, the role of the PINCHIILKICH-ILKBP complex in cell-matrix adhesion, spreading and fibronectin matrix assembly will be determined using reagents that modulate the complex formation. Third, PINCH-RP will be characterized and its potential role in the regulation of the assembly and functions of the PINCHIILKICH-ILKBP complex will be determined. These studies will provide important information on the assembly, function and regulation of the PINCH/ILK/CH-ILKBP complex and will lead to a better understanding of the general mechanism by which cells regulate cell adhesion and matrix assembly, and consequentially, a better understanding of the molecular basis underlying the pathogenesis of diseases associated with abnormal cell adhesion and matrix assembly.